Type 1 diabetes mellitus, a major cause of childhood morbidity in the United States, is a defect of immunoregulation in which a T lymphocyte-mediated autoimmune response to pancreatic beta-cell antigens leads to islet cell destruction, insulin deficiency, and overt diabetes mellitus. Loss of tolerance to islet cell epitopes probably arises from multiple immune defects that culminate in an imbalance between diabetogenic autoreactive T cells and protective regulatory T cells (T reg). The normal immune system contains a subset of CD4+ T cells that actively regulate T cells with autoreactive potential. These regulatory T cells appear to represem a distinct CD4+ T cell subset (expressing CD25 and CTLA-4), however their ontogeny and mode of action remain poorly understood. Significantly, CD4+ CD25+ T reg cells are present with similar frequency in hmnans, rats, and mice, suggesting that the survival factors and biological activity of these cells are evolutionarily conserved. Our central hypothesis is that Type 1 diabetic patients have a defect in the number or function of T reg cells specific for islet antigens. Such a net decrease in T reg activity enables autoreactive T cells to escape regulation. Current studies of T regs in autoimmune disease are hindered by the lack of organ/antigen-specific functional assays for T regs and lineage-specific T reg phenotypic markers. This proposal seeks to define the status of T reg activity in the context of the human autoimmune disease Type 1 Diabetes Mellitus (IDDM). We will investigate T reg number and function (Aim 1), T reg cell-surface phenotype (Aim 2) and the Notch signal pathway in T reg activity (Aim 3) in healthy and diabetic individuals. This research project is central to the objectives of the multi-project program. The mechanism of regulation of immune responses to self-antigen is likely to similar in many autoimmune diseases. As yet, little is known of T reg activity in human autoimmune disease. This project establishes methods for the analysis of regulatory T cell function on a global scale and on an antigen-specific basis. Such methods will be utilized by the other two basic research projects in the context of lupus and multiple sclerosis. The ability to monitor and manipulate suppressive function in autoimmune patients, and individuals predisposed to autoimmune disease, will be an important tool in the creation of the next generation of therapeutics.